Pharmacotherapy alleviates pathological changes in human direct reprogrammed neuronal cell model of myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) is a trinucleotide repeat disorder affecting multiple organs. However, most of the research is focused on studying and treating its muscular symptoms. On the other hand, despite the significant impact of the neurological symptoms on patients’ quality of life, no drug therapy was studied due to insufficient reproducibility in DM1 brain-specific animal models. To establish DM1 neuronal model, human skin fibroblasts were directly converted into neurons by using lentivirus expressing small hairpin RNA (shRNA) against poly-pyrimidine tract binding protein (PTBP). We found faster degeneration in DM1 human induced neurons (DM1 hiNeurons) compared to control human induced neurons (ctrl hiNeurons), represented by lower viability from 10 days post viral-infection (DPI) and abnormal axonal growth at 15 DPI. Nuclear RNA foci were present in most of DM1 hiNeurons at 10 DPI. Furthermore, DM1 hiNeurons modelled aberrant splicing of MBNL1 and 2, MAPT, CSNK1D and MPRIP at 10 DPI. We tested two drugs that were shown to be effective for DM1 in non-neuronal model and found that treatment of DM1 hiNeurons with 100 nM or 200 nM actinomycin D (ACT) for 24 h resulted in more than 50% reduction in the number of RNA foci per nucleus in a dose dependent manner, with 16.5% reduction in the number of nuclei containing RNA foci at 200 nM and treatment with erythromycin at 35 μM or 65 μM for 48 h rescued mis-splicing of MBNL1 exon 5 and MBNL 2 exons 5 and 8 up to 17.5%, 10% and 8.5%, respectively. Moreover, erythromycin rescued the aberrant splicing of MAPT exon 2, CSNK1D exon 9 and MPRIP exon 9 to a maximum of 46.4%, 30.7% and 19.9%, respectively. These results prove that our model is a promising tool for detailed pathogenetic examination and novel drug screening for the nervous system.


Introduction
Myotonic dystrophy type 1 (DM1) is an autosomal dominantly inherited multi-organ disease characterized by myotonia, muscle weakness, cataract, respiratory failure, cardiac abnormalities, endocrine and gastrointestinal dysfunction and neurological disturbance [1]. It is the most common form of adult muscular dystrophy with an estimated prevalence of 1:8000 [2]. DM1 neurological manifestations include cognitive dysfunction, memory impairment, emotional deficiency, apathy, mental disorders and excessive daytime sleepiness [3]. Imaging techniques demonstrated cortical gray matter atrophy in frontal, parietal, temporal and occipital regions with diffused white matter lesions and ventricle dilatation in DM1 brain [4][5][6].
It is postulated that myopathy and neuropathological changes in DM1 brain is caused by CTG trinucleotide expansion in the 3 ' untranslated region of dystrophia myotonica protein kinase (DMPK) gene located on chromosome 19 and a repeat size of 50 or more is considered pathogenic in DM1. After transcription, the expanded CUG repeats are entrapped in the nucleus leading to the formation of hairpin loops known as nuclear RNA foci which sequester muscle-blind like (MBNL) family proteins responsible for alternative splicing of other genes. Thus, resulting in many mis-splicing events [7][8][9][10][11]. Repeat-associated non-ATG (RAN) translation is another pathological mechanism studied in DM1 where the expanded CUG and CAG repeats in sense and antisense transcripts, respectively, form hairpin loops leading to translation of toxic proteins without an ATG start codon [12].
Several therapeutic approaches have been used for the treatment of DM1 muscular symptoms including: the use of antisense oligonucleotide [13], reduction of CUG transcript level [14], inhibition of MBNL1 sequestration [15][16][17], cleavage of CUG expanded repeats by CRISPR/CAS9 editing [18] or small molecules [19] and blocking the production of CUG repeats by genome modification [20]. However, drug screening for treatments targeting neurological symptoms is lagging behind due to the lack of a good neuronal model. Although animal models were found to recapitulate many DM1 phenotypes in post-mortem brain tissue, they could not fully recapitulate neuropathological features of DM1. For example, discrepancies in regulating alternative splicing of exons were observed in DM1 animal models. This is mostly attributed to species differences between animals and humans [11,21,22]. Also, development of phenotypic brain abnormalities in DM1 animal models takes several months (axonal and dendrites degeneration was observed at 9 months and aberrant splicing at 12 months) [23]. Therefore, it makes drug screening a time-consuming process. Brain autopsies can provide insight into brain pathological changes, yet it is limited by other confounders, such as disease severity or comorbidities by the time of death and it cannot be used for drug screening. Obtaining brain biopsy from living patients is impractical and difficult [24]. In vitro modelling using induced pluripotent stem cells (iPSC) derived neural stem cells provides a valuable tool for studying pathological changes in patients, as well as for treatment development. Nevertheless, direct reprogramming of patients' fibroblasts into neurons skipping the pluripotent phase provides a good opportunity for modelling neuropathological changes and for drug screening concomitantly with the additional advantage of time and cost saving as compared to iPSC derived neural stem cells. Moreover, in contrast to iPSC derived neural stem cells which revert to their pluripotent state losing their epigenetic signature, induced neurons by direct reprogramming can maintain their epigenetic changes providing an additional advantage for investigating age related diseases like neurodegenerative diseases [25].
Neurons generated by direct reprogramming of human fibroblasts are known as human induced neurons (hiNeurons). Direct reprogramming of patients' fibroblasts into neurons was successful in modelling some neurological diseases such as ALS [26], Huntington's disease [27], Alzheimer's disease [28] and Parkinson's disease [29]. Trans differentiation of fibroblasts

PLOS ONE
In vitro modelling of myotonic dystrophy type 1 human neurons by direct reprogramming of patients fibroblasts PLOS ONE | https://doi.org/10.1371/journal.pone.0269683 July 1, 2022 2 / 26 no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests:
The authors have read the journal's policy and have the following competing interests: This article discusses neuropathological changes treatment only but the patent is related to efficacy of erythromycin in treating DM1 muscular symptoms and pathological changes; Title (Agent for treatment of myotonic dystrophy) No.
(WO2017010382A1) Priority to JP2015140599). This does not alter our adherence to PLOS ONE policies on sharing data and materials.
into neurons was achieved by using a combination of transcription factors and NeuroD1 [30], expression of miR-9/9 � and miR-124 combined with Ascl1 and Myt1l transcriptional factors and NeuroD2 [31], or by chemical cocktail [28]. Also, induced neurons were generated from mouse fibroblasts by downregulation of poly-pyrimidine tract binding protein (PTBP) which occurs during normal brain development. This was achieved by using small hairpin RNA against PTBP (shPTBP) which resulted in the collective induction of miR-124 and miR-9, five transcription factors (Ascl1, Brn2, Myt1l, Zic1, and Olig2) and NeuroD1, all of which synergizes with each other to enhance conversion of fibroblasts into neurons. Induced neurons generated by this method were found to be functional with glutamatergic and GABAergic responses [32]. Since glutamate and GABA neurotransmitters are utilized by cortical projection neurons and cortical interneurons, respectively [33], we thought this method of direct reprogramming will be of importance in modelling DM1 cortical neurons since they are affected in this disease as proved by previous studies [8,34].
In the present study, we are using direct reprogramming approach for the first time to generate DM1 human neurons from patients' fibroblasts to model and effectively treat the phenotypic brain abnormalities and aberrant splicing of gene transcripts reported previously in the literature by drug therapy. Our DM1 hiNeurons successfully recapitulated neurodegeneration represented by their lower viability and abnormal axonal outgrowth compared to ctrl hiNeurons. We also found accumulation of nuclear RNA foci (one of the molecular hallmarks of DM1) in DM1 hiNeurons where their treatment with actinomycin D was effective in reducing the number of RNA foci. Additionally, DM1 hiNeurons modelled some aberrant splicing events observed in DM1 brain which were rescued by erythromycin treatment; hence, emphasizing the utility of this model for investigating neuropathological mechanisms and its applicability for drug screening studies.

Cell culture and direct conversion of fibroblasts into neurons
Healthy and DM1 human skin fibroblasts were purchased from the Coriell Institute for Medical Research (Camden, NJ, USA). Further details are provided in Table 1. Fibroblasts were seeded into 8 wells Lab-Tek II chambered glass slide (Cat# 154534, Nunc), 24-well or 6-well plates (Corning Incorporated Costar) at a density of 2 x 10 4 , 3 x 10 4 or 1.2 x 10 5 cells, respectively. Chamber glass was coated with 2.5 μg/mL laminin (Cat# 120-05751, FUJIFILM Wako Pure Chemical Corporation) plus 0.15 mg/mL PLL in PBS for at least 40 min before seeding whereas 24-well and 6-well plates were coated with 0.1mg/mL gelatin (Cat# G1890, Sigma) in distilled water.

Viability study
To compare viability differences over time between ctrl and DM1 hiNeurons, live cell imaging was performed using the bright field/phase of KEYENCE BZ-X710 all in one fluorescence microscope with 10x magnification at the following intervals: 8, 10, 12, 16, 22 and 28 DPI. Each 10 images captured from the top to the end of the culture well represent 1 collection for a total of 4 collections from right to left. For each time point, a total of 10 images per sample replicate from 4 different collections (30 images per sample from three replicates) were included in the analysis to insure good representation of neuronal viability in the whole well. Neurons were considered viable, and thus included in the analysis when they were adherent and exhibited neuronal morphology of expanded cell bodies and projecting neurites regardless of their neurites' lengths whereas cells clumping together and floating in the medium were considered dead and so excluded from the analysis. Viable cell counting was performed manually using ImageJ (RRID:SCR_003070, The National Institute of Health).

Axonal degeneration analysis
Ctrl and DM1 human fibroblasts were cultured in 48 wells and converted into hiNeurons by direct reprogramming. At 15 DPI, ctrl and DM1 hiNeurons were co-immunostained with TUJ1 and an axon specific marker (SMI-312) as previously described. Images were captured by KEYENCE BZ-X710 all in one fluorescence microscope at 10x magnification and axonal length measurement was performed using Simple Neurite Tracer plugin provided by ImageJ (The National Institute of Health). Measurements were taken from the end of neuronal soma to the end of axonal shaft. The longest, medium and shortest axons from each photo were included in the analysis. A total of 36 images for each sample (from three replicates) were analyzed, each including measurements of 3 axons by the defined criteria (total 108 axons/ sample).

Cytotoxicity study
To test the tolerability of DM1 and ctrl hiNeurons to actinomycin D (Cat# A9415, Sigma) and erythromycin lactobionate (CAS Number# 3847-29-8), fibroblasts were seeded into 96-well plate at a density of 17.5 x 10 3 cells and converted into neurons by direct reprogramming. At 8 DPI, concentrations ranging from 25-300 μM of erythromycin were incubated with hiNeurons for 48 h and tolerability of cells was assessed visually using Eclipse Ts2 microscope (Nikon) at 4x magnification. Poor tolerability was defined as increased cellular clumping and floating in the culture medium. The same procedure was applied for ACT except that neurons were incubated at 9 DPI with concentrations ranging from 5-100 nM for 24 h. After reconstitution of erythromycin and ACT, the required drug concentrations were achieved by diluting the drugs in neuronal medium. For comparison, the same amount of neuronal medium without drug was added as placebo.

Fluorescence in situ hybridization/ Immunocytochemistry
Visualization of RNA foci was done as previously described [36,37]. DM1 and ctrl hiNeurons were maintained until 10 DPI in 8 wells Lab-Tek II chambered glass slide and washed once with PBS for 1 min, followed by fixation with 3% paraformaldehyde in PBS for 15 min at room temperature. Then, cells were washed with PBS twice for 5 min each and permeabilized by adding 0.5% Triton X-100 (Cat# 35501-15, Nacalai Tesque) in PBS for 5 min. A pre-hybridization solution consisting of 30% formamide (Cat# 16229-95, Nacalai Tesque) with 2x SCC (Cat# 15557-044, Invitrogen) in distilled water was added on cells for 10 min. The following procedures were carried out while protecting from light. Cells were hybridized by adding 1 ng/ μL of 5' Texas red 2-O-methyl-CAG RNA probe (CAGCAGCAGCAGCAGCAGCA) (IDT) in hybridization buffer (33% formamide, 2x SCC, 2 mM vanadyl complex (Cat# S1402S, New England Biolabs), 0.02% BSA (Cat# 2320, Takara), 70 μg/mL yeast tRNA (Cat# AM7119, Thermo Fisher Scientific) in distilled water) and placed in humidified chamber at 37˚C for 2 h. At the same time, the pre-hybridization solution was placed in hybridization chamber for 2 h at 42˚C after which is called post-hybridization solution. Then, cells were washed by adding post-hybridization solution and placed in hybridization chamber for 30 min at 42˚C followed by 1 time wash with 1x SCC for 30 min and additional wash with PBS for 5 min. Thereafter, immunostaining of neuronal cells with TUJ1 antibody was performed as previously described. After completion of immunostaining, chamber was separated from the glass slide and DAPI was added to visualize nuclei before sealing the glass slide. Images for aggregation of RNA foci in neurons were captured at 20x magnification by KEYENCE BZ-X710 all in one fluorescence microscope and the number of nuclear or cytoplasmic RNA foci was counted manually using Keyence BZ-X Analyzer software (version 1.3.1.1). 108 neurons present in 6 different fields were analyzed for each sample replicate (total is 324 neurons for each sample from 3 replicates).
As for treatment study, ACT at a concentration of 100 or 200 nM was added to DM1 hiNeurons at 9 DPI for 24 h followed by the same procedure.
The following equation was used to calculate percentage of nuclear or cytoplasmic RNA foci reduction relative to placebo (P) treated hiNeurons: % RNA foci reduction ¼

RNA extraction, RT-PCR and alternative splicing analysis
For comparison between ctrl and DM1 hiNeurons alternative splicing pattern, hiNeurons were maintained until 10 DPI, washed once with PBS and RNA was extracted using Micro RNA Extraction kit (Cat# 74004, Qiagen) with DNase1 treatment following manufacturer protocol. About 73 ng of RNA from each sample was reverse transcribed simultaneously into cDNA using random hexamers and Superscript IV First Strand Synthesis System (Cat# 18091050, Invitrogen) according to manufacturer's recommendations. Desired transcripts were amplified by PCR using 0.3 μM primers flanking exons of interest (See S1 Table) and AmpliTaq Gold 360 Master Mix (Cat# 4398881, Applied Biosystems). The amount of cDNA template constituted 7.5% of the total amplification reaction volume. The following PCR conditions were applied: activation of AmpliTaq Gold 360 Master Mix for 10 min at 95˚C, followed by 31, 33 or 35 cycles (as indicated in S1 Table) of denaturation for 30 seconds at 95˚C, annealing for 30 seconds at the temperature specified in S1 Table for each transcript and extension for 1min at 72˚C and 1 cycle of a final extension for 7 min at 72˚C.
Amplified PCR products were analyzed by MultiNA automated microchip electrophoresis system (MCE-202 MultiNA, Shimadzu Manufacturing Co., Ltd.) using DNA-500 kit (Cat# P/ N 292-27910-91, Shimadzu). The results were viewed as electropherogram peaks and area under the peak was quantified automatically by MultiNA viewer software.
To calculate percentage exon inclusion (% exon inclusion), the area under the peak (AUC) of exon inclusion isoform was divided by the total area of the peaks.
Rescuing of mis-splicing study At 8 DPI, ctrl and DM1 hiNeurons were incubated with 35 or 65 μM erythromycin or placebo for 48 h simultaneously and for each sample about 100 ng of the extracted RNA by the previously explained procedure was simultaneously reverse transcribed into cDNA. After calculating % exon inclusion, the change percentage (% change) relative to placebo (P) treated hiNeurons was calculated as follows: While rescue percentage (% rescue) was calculated as previously described [17].

Statistical analysis
Three DM1 and three apparently healthy biological replicates (samples) were used to compare various measures between DM1 and control groups. For each sample, multiple cells, nuclei or axons were counted/measured (as indicated in each figure legend) and then averaged to obtain a single value. Two-tailed unpaired t-test was used to compare between the means of DM1 and control groups. For treatment studies (reduction of RNA foci and rescuing of mis-splicing), repeated measures one-way ANOVA test without multiplicity adjustments was used to compare between the means of three groups: placebo, low or high concentration of the tested drugs. Parametric tests were used based on the assumption of normality as it is not applicable to use normality tests to assess this assumption when sample size is small.
To confirm if results of the aberrant splicing treatment experiments were robust, we applied three approaches: 1) by using repeated measures ANOVA to compare between exon inclusion percentage values (raw data), 2) by using repeated measures ANOVA to compare between the log values of exon inclusion percentage or 3) by using repeated measures ANOVA to compare between the percentage of change values instead of exon inclusion percentage values. The three approaches proved statistical significance; thus, p values of the first approach were reported because it depends on raw data and resembled the results of at least one of the other selected approaches.
Experiments were performed in triplicate. Analysis was performed by GraphPad Prism9 (RRID: SCR_002798, GraphPad Software Inc) and the results were considered statistically significant when p<0.05.
To generate hiNeurons, healthy and DM1 fibroblasts were transduced with lentivirus coexpressing shPTBP and puromycin resistant gene [32]. Converted neurons were selected by adding puromycin for 2 days. After that, cells were maintained in neuronal medium and at 6 DPI, fibroblasts started to acquire rounded cell bodies, however neurites were still undefined clearly. Since the viability of neurons was poor by 8 DPI in ctrl and DM1 hiNeurons, we decided to add 2-mercaptoethanl and sodium pyruvate as they were shown to improve maintenance of neurons for a longer term [38][39][40]. Indeed, the viability of neurons have improved at 8 DPI and cells exhibited neuronal like morphology with defined processes. To optimize their development, neuronal growth factors (BDNF, GDNF, NT3 and CNTF) were added to neuronal medium and by 10 DPI, we noticed neuronal like cells with expanded cell bodies and extended neurites. The majority of neurons were bipolar with some multipolar neurons (Fig 1a). Most of these cells showed positive immunoreactivity with TUJ1 anti-body which recognizes the neuronal specific class III β-tubulin antigen [41], and thus confirming successful conversion of fibroblasts into neurons to a similar extent in both groups (Fig 1b and 1c). About 5% of hiNeurons were positive for the mature neuronal marker microtubule-associated protein 2 (MAP2) [42] (Fig 1b and 1d). We confirmed that hiNeurons were composed of a mixture of inhibitory and excitatory neurons as they displayed markers of GABAergic and glutamatergic neurons from 10 DPI when immunostained with gamma-aminobutyric acid (GABA) and glutamate antibodies, respectively (Fig 1e). At 15 DPI, some hiNeurons expressed neuronal nuclear antigen (NeuN) which is a useful marker for assessment of neuronal maturation [43] (Fig 1e). Nevertheless, no synaptic connections were formed between hiNeurons when investigated at 10 or even 17 DPI by immunostaining with synapsin 1 antibody (SYN1) (Fig 1e).
Ctrl and DM1 hiNeurons are composed of a mixture of inhibitory and excitatory neurons as proved by the positive immunoreactivity for GABA and glutamate from 10 DPI. Thus, indicating that this model was successful in generating cortical neurons, the type of neurons that are mostly affected in DM1 [8,34]. However, this model, showed a small percentage of mature neurons at 10DPI without any synaptic connections. Collectively, these results show that this model is applicable for phenotypic or biochemical studies, however, more optimization will be needed before considering this model for neurophysiological analysis.

Reduced viability of DM1 hiNeurons
Neuronal loss in frontal and parietal cortices with intensive loss in occipital cortex was reported in DM1 postmortem brain [34]. To investigate if there are any viability differences between ctrl hiNeurons and DM1 hiNeurons over a period of 4 weeks, the same number of ctrl and DM1 human fibroblasts were cultured in 24 wells and converted into hiNeurons using the same lentivirus stock.
We selected 8 DPI as the baseline count of viable neurons in each group because it is the day at which converted cells acquired neuronal like morphology. Viability was monitored at the following intervals: 10, 12, 16, 22 and 28 DPI. We considered neurons as viable and included them in the analysis when they were adherent and exhibited neuronal morphology of expanded cell bodies and projecting neurites regardless of their neurites' lengths whereas cells clumping together and floating in the medium were considered dead and so excluded from the analysis.
The decreased viability observed over time in both groups indicates that most cells are successfully converted into neuronal like cells which were not actively proliferating unlike fibroblasts that are known to proliferate overtime.

Abnormal axonal outgrowth in DM1 hiNeurons
To verify whether the faster loss of DM1 hiNeurons is associated with any axonal defects, fibroblasts for all cell lines were cultured in 48 wells and the same direct reprogramming protocol was applied. Since we observed a big difference between the viability of ctrl hiNeurons and DM1 hiNeurons between 12 & 16 DPI, we expected that a difference between the two groups may be evident at any time point included in this timeframe. Accordingly, hiNeurons were coimmunostained with SMI-312, a mixture of monoclonal antibodies targeting highly phosphorylated axonal epitopes on neurofilaments M and H [44], and TUJ1 at 15 DPI. The length of axons showing positive immunoreactivity for the axon specific marker (SMI-312) was By 15 DPI, the mean of the longest axons measured in DM1 hiNeurons was 221.5 μm compared to 457.9 μm in ctrl hiNeurons (p = 0.0090) while that of the shortest was 25.9 μm in DM1 vs 44.7 μm in ctrl hiNeurons (p = 0.0093). Overall, the axonal length of DM1 hiNeurons was shorter than ctrl hiNeurons with an average length of 119.6 μm in DM1 hiNeurons compared to 247.0 μm in ctrl hiNeurons (p = 0.0058) (Fig 3a-3e).

Accumulation of nuclear RNA foci in DM1 hiNeurons
To confirm the accumulation of expanded CUG repeats as RNA foci in the nuclei of DM1 hiNeurons, fibroblasts of controls and DM1 patients were cultured in chambered glass slide and induced into neurons by the same protocol. At 10 DPI, fluorescence in situ hybridization (FISH) was performed using 5' Texas red 2-O-methyl-CAG RNA probe followed by immunostaining with TUJ1 and DAPI.
Results confirmed the presence of nuclear RNA foci in most of DM1 hiNeurons in contrast to the negligible presence of RNA foci in controls (Fig 4a1, 4a2 and 4b). The average count of RNA foci per nucleus in DM1 hiNeurons was 3.82 whereas the average observed in their counterparts was 0.007 (p = 0.0008) (Fig 4c). Furthermore, RNA foci were also present in the cytoplasm of DM1 hiNeurons (Fig 4a2). However, cytoplasmic RNA foci were fewer than nuclear RNA foci with an average of 0.64 cytoplasmic RNA foci per DM1 hiNeuron (p = 0.0199) (S1a Fig). Thus, this model was successful in recapitulating accumulation of nuclear RNA foci in DM1 neurons, a neuropathological feature observed in DM1 post-mortem brain tissue [8] and animal [23] and iPSC derived neural stem cells models [20]. Although cytoplasmic RNA foci were not reported in DM1 brain, their presence in this model could be attributed to the existence of cytoplasmic RNA foci in DM1 fibroblasts, the origin from which hiNeurons are derived, as reported previously [46]. Nonetheless, it was found that cytoplasmic RNA foci are not sufficient to provoke DM1 pathological features [46].

Treatment by actinomycin D reduces nuclear RNA foci in DM1 hiNeurons
To test if nuclear RNA foci in DM1 hiNeurons can be abolished or decreased with drug therapy, we thought to use actinomycin D (ACT) also known as dactinomycin, a drug that was previously reported to reduce the number of RNA foci per nucleus by 50% in DM1 HeLa cell model when added at a concentration of 10 nM for 18 hours [14].
Accordingly, several concentrations up to 100 nM of ACT were initially tested for 24 h to dissolve or reduce nuclear RNA foci in DM1 hiNeurons by 10 DPI. Surprisingly, the previously reported concentration of 10 nM did not show any effect in DM1 hiNeurons but a concentration of 100 nM was effective in reducing RNA foci. To determine if a higher concentration will be more effective, we compared the effect of ACT treatment at 100 nM with 200 nM for 24 h. FISH study at 10 DPI followed by immunostaining revealed that 100 nM of ACT was effective in reducing the number of RNA foci per nucleus by 56% while 66% reduction was achieved by doubling the concentration (p<0.0001 for both concentrations). The mean effect difference between the two concentrations was also significant (p = 0.0256) (Fig 4a3, 4a4, 4d and 4e). Furthermore, treatment with 200 nM ACT reduced the number of nuclei containing RNA foci by 16.5% (p = 0.0199) (Fig 4f). Although some cellular toxicity was observed at 200 nM, it was not severe to exclude its use in the treatment study as increased cellular death is expected with chemo-therapeutic agents like ACT [47] (S2 Fig). Also, treatment of DM1 hiNeurons with 100 nM or 200 nM ACT reduced cytoplasmic RNA foci by 15.75% or 46.85%, respectively. However, the results were statistically insignificant (p = 0.1500, p = 0.0651, respectively) (S1b-S1d Fig). Our results showed dose dependent effect of ACT on the treatment of DM1 hiNeurons, however, it was difficult to achieve complete dissolution of nuclear RNA foci.

Dysregulated alternative splicing in DM1 hiNeurons
To explore whether DM1 hiNeurons can model abnormal splicing events previously observed in DM1 post-mortem brain, RNA was extracted from DM1 and ctrl hiNeurons at 10 DPI followed by simultaneous synthesis of cDNA for all samples by reverse transcriptase-polymerase chain reaction (RT-PCR). Then, the desired gene transcripts were amplified using previously published primers flanking exons of interest (See S1 Table). PCR products were analyzed by MultiNA automated microchip electrophoresis system and percentage of exon inclusion was calculated to compare inclusion of alternatively spliced exons between the two groups.
We found preferential inclusion of exon 5 of MBNL 1 and 2 (p = 0.0010 and p = 0.0001, respectively), as well as increased inclusion of exon 8 of MBNL2 in DM1 hiNeurons (p = 0.0060) (Fig 5a and 5b). These results are in accordance with the previously published data in DM1 post-mortem brain tissue [10,48]. Since our model was successful in recapitulating the aberrant splicing of MBNL1 and 2 which are known to regulate alternative splicing of other genes, we further investigated alternative splicing of transcripts, specifically those involved in neurite outgrowth as our previous results indicated abnormal axonal extension in DM1 hiNeurons. Accordingly, alternative splicing of microtubule associated protein tau (MAPT), myosin phosphatase Rho interacting protein (MPRIP) and casein kinase 1 delta (CSNK1D) was examined.
Collectively, these results demonstrate the success of DM1 hiNeurons in modelling deregulated alternative splicing described previously. Moreover, the deregulated alternative splicing of MBNL1 and 2 resulted in mis-splicing of their dependent transcripts.

Treatment of DM1 hiNeurons by erythromycin lactobionate rescues missplicing of MBNL1, MBNL2 and their dependent transcripts
Previous studies have shown that treatment of DM1 fibroblasts and myotubes by erythromycin lactobionate could rescue aberrant splicing of MBNL 1 and 2 exon 5 and the splicing of other MBNL dependent transcripts in a dose dependent manner [16,17].
To verify whether similar results can be obtained in DM1 hiNeurons, we tested treatment with several concentrations of erythromycin lactobionate. Our treatment trial showed that treatment with 65 μM erythromycin can improve mis-splicing of MAPT exon 2 whereas treatment with a higher concentration of 100 μM was not. Accordingly, DM1 hiNeurons were simultaneously treated with 35 μM or 65 μM erythromycin or placebo for 48 h and RNA was extracted at 10 DPI followed by the same pre-mentioned procedures for alternative splicing hiNeurons. Each sample is presented in different color. Each symbol represents the percentage of nuclei containing RNA foci for each sample replicate. Line represents the mean. ACT treatment reduced the percentage of nuclei with RNA foci by 10.3% or 16.5% at 100 nM or 200 nM ACT, respectively. Counting was performed manually. n = 3 for each group, a total of 324 nuclei were analyzed per sample. ���� P<0.0001, ��� P<0.001, �� P<0.01, � P<0.05 and ns, not significant compared by repeated measures one-way ANOVA test. P, placebo (same amount of diluent without drug).
To ensure that the studied doses are not associated with adverse effects, the same concentrations of erythromycin were studied on ctrl hiNeurons and no significant changes were observed on alternative splicing of any of the studied transcripts (Figs 6a-6c and 7a-7c).
These findings confirm that erythromycin treatment was effective in correcting mis-splicing of all studied transcripts. Importantly, no pronounced cellular toxicity was observed at 35 μM but the additional improvement achieved with the higher concentration is accompanied with some additional risk of cytotoxicity (S3 Fig).

Discussion
Unravelling the underlying neuropathological mechanisms of DM1 and targeting them with therapeutic interventions is essential to improve the quality of life of DM1 patients. Although, DM1 is a multi-organ disease, most of the research is directed toward studying the underlying pathomechanisms of myopathy and based on this knowledge many therapeutic approaches have been employed for the treatment of its muscular symptoms. This could be explained by the availability of animal and in vitro models capable of recapitulating the muscular symptoms. On the other hand, no drug therapy has been studied for the treatment of neurological abnormalities due to insufficient reproducibility in DM1 brain-specific animal models as alternative splicing differences were observed in them. Also, using iPSC derived neural cells for drug screening is limited by its costly and longer procedure. Therefore, the need arises to establish a new in vitro neuronal model.
In this study, direct reprogramming of DM1 patients' fibroblasts into neurons by the stable knockdown of PTBP was used to generate DM1 in vitro neuronal model. DM1 hiNeurons recapitulated features of decreased viability, abnormal axonal outgrowth, nuclear RNA foci accumulation and alternative splicing abnormalities observed in DM1 post-mortem brain tissues and in vitro and animal models. In our study, aberrant splicing of gene transcripts involved in neurite outgrowth preceded axonal outgrowth defects. Furthermore, treatment of DM1 hiNeurons with ACT significantly reduced the number of nuclear RNA foci by more than 50% and treatment with erythromycin rescued mis-splicing of MBNL1 exon 5 and MBNL2 exons 5 and 8 up to 17.5%, 10% and 8.5%, respectively. Furthermore, erythromycin rescued the aberrant splicing of MAPT exon 2, CSNK1D exon 9 and MPRIP exon 9 to a maximum of 46.4%, 30.7% and 19.9%, respectively. Importantly, the dual studies of viability and measurement of axonal length where rapid decline in viability and abnormal axonal outgrowth were observed in DM1 hiNeurons, indicate that DM1 neurons undergo faster degeneration than ctrl neurons. These results are in accordance with the neurodegeneration phenomenon observed over a decade in DM1 patients [45], and thus explain the finding of cerebral atrophy in DM1 postmortem brain.
DM1 hiNeurons modelled aggregation of nuclear RNA foci and treatment with 100 or 200 nM ACT for 24 h reduced the number of RNA foci per nucleus by 56% or 66%, respectively, with 16.5% reduction in the number of nuclei containing RNA foci by the higher concentration. Similar to the results observed in DM1 HeLa cell model after treatment with 10 nM ACT for 18 h [14]. Both findings indicate the difficulty of complete dissolution of RNA foci by ACT in DM1 in vitro models. In this study, using 10x higher concentrations of ACT to achieve similar results, suggests that neurons are insensitive to low concentrations of ACT. The need for higher doses in non-dividing cells like neurons may be explained by the pharmacodynamic properties of ACT as it is a cell-cycle specific drug where cells in the G 1 -S border phase are most sensitive [51].
DM1 hiNeurons were successful in modelling aberrant splicing of MBNL1 exon 5 and MBNL2 exons 5 and 8, MAPT exon 2, CSNK1D exon 9 and MPRIP exon 9 as observed in postmortem DM1 brain but to a lesser extent than what was previously reported for the last three mentioned transcripts [11,22,48,50]. This may be explained by the following reasons: 1) DM1 hiNeurons model current neuropathological changes in DM1 which may be progressively altered at the time of death. 2) The brain is composed of variety of cells. For example, RNA foci were also found in oligodendrocytes of DM1 brain [8] but whether splicing defects occur in these cells is yet unknown. Furthermore, the expression level of tau in oligodendrocytes and astrocytes in DM1 needs further investigation as it was found to play a role in impairment of neuronal network and the spread of pathological tau in a number of neurodegenerative diseases like Alzheimer's disease, progressive supranuclear palsy and Pick's disease [52][53][54]. 3) DM1 hiNeurons are cortical neurons not specified to any brain region and it was shown that neurons obtained from different brain regions from the same patients show some variation in the extent of mis-splicing [55]. Likewise, discrepancies in splicing abnormalities between grey matter and white matter in the same patients have been reported [48]. 4) Individual differences of disease severity. 5) Small sample size in this study. Nevertheless, the differences between ctrl and DM1 hiNeurons remain to be significant; thus, supporting the applicability of this model for drug screening studies.
It was found that preferential inclusion of exon 5 of MBNL1 and 2 influence the localization of MBNL proteins in the nucleus and increased inclusion of MBNL2 exon 8 is associated with reduced exchange of MBNL proteins between the nucleoplasm and expanded CUG hairpin loops; thus, stabilizing the CUG-MBNL complex [56]. Tau protein is involved in stabilization and assembly of microtubules, transport of vesicles and organelles on microtubules and regulation of cell shape and motility [57][58][59], and hence play an important role in the maintenance of axonal structure and function. Suppression of tau by antisense oligonucleotide was shown to reduce axonal length in cerebellar neurons compared to sense treated neurons [60]. CSNK1D is involved in neurite outgrowth, circadian rhythm generation and Fas-mediated apoptosis [61,62]. The relationship between CSNK1D and neurodegeneration has been proven in amyotrophic lateral sclerosis where a pharmacological inhibition of CK1-D (the enzyme encoded by CSNK1D) resulted in decreased phosphorylation of TDP-43 (TAR DNA-binding protein 43) by this kinase and enhanced preservation of spinal motor neurons [63]. MPRIP modulates neurite outgrowth through inactivation of RhoA-Rho kinase (ROCK) actomyosin pathway that signals growth cone collapse and neurite retraction [64]. MPRIP mis-splicing can negatively affects its role in promoting neurite outgrowth. For example, PARK2 knockout human iPSC derived neural cells, a model of Parkinson's disease, showed that increased Rho signaling was accompanied by decreased levels of phospho-MPRIP, impaired neurite outgrowth and increased cell migration whereas pharmacological inhibition of RhoA signaling in this model resulted in improved neurite outgrowth and decreased cell migration [65].
The higher concentration of erythromycin was associated with more rescue of the dysregulated splicing in DM1 hiNeurons than the lower concentration. These data are in concordance with the dose-dependent response observed in DM1 human fibroblasts, human derived myotubes and animal models [16,17]. Erythromycin was significantly effective in rescuing MBNL1 exon 5 and MBNL2 exons 5 and 8 with a parallel rescue of their dependent transcripts: MAPT, CSNK1D and MPRIP. In vitro study found that MBNL1 loss induced MAPT exon 2 exclusion [10]. To the contrary, single knockout of MBNL1 in animal model could not induce mis-splicing of MAPT exon 2 but single knockout of MBNL2 or double knockout of MBNL1 and 2 induced skipping of MAPT exon 2 [11,22]. In our study, correction of MAPT exon 2 missplicing might be the result of the parallel rescue of MBNL1, MBNL2 or a cumulative result of the correction of both mis-splicing events. MBNL2 is the main regulator of CSNK1D alternative splicing as association between depletion of MBNL2 and decreased inclusion of CSNK1D exon 9 was proved by in vitro and animal studies [11,21]. Moreover, exon 8 of MBNL2 was found to be the strongest regulator of CSNK1D alternative splicing in DM1 HeLa model [56]. Hence, correction of CSNK1D exon 9 dysregulated splicing can be attributed to the rescue of MBNL2 by erythromycin treatment. Aberrant splicing of MPRIP exon 9 was also rescued by erythromycin treatment. Previous data showed a lack of significant splicing alteration of MPRIP exon 9 in MBNL1 knockout mice model [22]. Therefore, its correction is perhaps a ramification of MBNL2 mis-splicing rescue by erythromycin.
Actinomycin D is an anti-neoplastic drug currently approved for the treatment of different types of cancer such as Wilms' tumor and Ewing sarcoma. Furthermore, a clinical study reported significant improvement in time to progression and overall survival of pediatric patients suffering from CNS atypical teratoid rhabdoid tumor when treated by chemotherapy regimen containing ACT [66]. Erythromycin is a macrolide antibiotic currently prescribed for the treatment of a variety of bacterial infections including acne, bronchitis and urethritis. Although erythromycin is not approved for CNS infections, some reports suggest its efficacy in treating CNS infections [67]. Importantly, the observed beneficial effects of ACT and erythromycin in DM1 results from interference with the underlying molecular pathology of the disease where ACT exerts its therapeutic effects by reducing CUG-RNA transcript level [14] whereas erythromycin by direct inhibition of MBNL1 sequestration via competitive binding to the expanded CUG repeats [16].

Conclusion
Overall, our results provide evidence that DM1 hiNeurons is a promising model for studying and investigating the underlying pathological mechanisms of DM1 brain and it will pave the way for future drug development.
In the future, we hope to employ this model for drug screening and for unravelling the correlation between DM1 splicing dysregulation and phenotypic abnormalities. Also, it will be interesting to explore if this model can reproduce pathological changes of other neurological and psychiatric diseases characterized by involvement of cortical neurons such as multiple sclerosis [68], corticobasal degeneration [69], autism spectrum disorders [70], among others.